Photo-control of metabolism

ABSTRACT

Disclosed are peptides represented by IVC 1 SLC 2 SC 3 TAW and C 1 SLC 2 SC 3  wherein I stands for isoleucine, V for valine, C 1  for cysteine, C 2  for cysteinesulfinic acid, C 3  for cysteinesulfenic acid, S for serine, L for leucine, T for threonine, A for alanine, and W for tryptophan. These peptides can impart photoreactivity to a protein by binding to a non-heme iron. Also disclosed are methods for imparting photoreactivity to cells by introducing the peptide sequences into at least one protein which is involved in a metabolic system and/or energy metabolic system of the cells. There can be provided peptide sequences with a shorter peptide chain capable of imparting photoreactivity, which can be easily introduced into a protein with a little risk in degrading original function of the protein. There are also provided methods enabling control of metabolic reactions by imparting photoreactivity to cells with the peptides.

FIELD OF THE INVENTION

[0001] The present invention relates to a peptide which can impartphotoreactivity to a protein, and a method for controlling metabolism ofcells by introducing a sequence of the peptide into at least one proteinwhich functions in a metabolic system so that the protein should acquirephotoreactivity, thereby controlling metabolism of cells.

BACKGROUND OF THE INVENTION

[0002] Nitrile hydratase (NHase) is an enzyme which is produced inmicroorganisms and converts a nitrile compound into an amide compound byhydration. It is a soluble metalloprotein containing iron or cobalt atomin its active center. NHase derived from Rhodococcus sp. N-771 strainhas a non-heme iron center of mononuclear low-spin six coordinateFe(III). NHases have been isolated from several kinds of bacterialcells, and all of them consist of two kinds of subunits, α and β. Bothof the subunits have a molecular weight of about 23,000. NHases ofRhodococcus sp. N-771, N-774, and R312 are considered to be the sameenzyme because their base sequences are identical to each another, andtheir enzymatic activity varies with light irradiation. Namely, whenbacterial cells exhibiting high activity are left in the dark, theenzyme activity is reduced, and the activity is increased again byphoto-irradiation.

[0003] It was recently revealed that photodissociation of nitric oxide(NO) which is bound to the non-heme iron center of inactive form Nhaseactivates the enzyme (Odaka et al., J. Am. Chem. Soc., 119, 3785-3791(1997)). However, because the structure of the non-heme iron centerwhich is the photoreactive site was not elucidated yet, the detailedmechanism of the photoreaction remained unclear. Therefore, the presentinventors performed structural analysis of the non-heme iron center, andreported the results (for example, see Protein, Nucleic acid, Enzyme,Vol.42, No.2, p38-45 (1997)). That is, the present inventor isolated theα and β subunits from the inactive form enzyme under denaturationcondition, and found that the NO-binding type non-heme iron center ispresent on the α subunit. Therefore, they further performed enzymaticdegradation of the α subunit with trypsin, and purified the resultingpeptides by reversed phase chromatography under a neutral condition. Asa result, a peptide consisting of 24 residues of ₁₀₅N to ₁₂₈K bindingone iron atom and NO has been isolated. This region was well conservedin various kinds of NHases, and contained a cysteine cluster predictedto be a metal-binding site (₁₀₉C-S-L-₁₁₂C-S-₁₁₄C).

[0004] By the way, development of processes for producing usefulbiomolecules such as amino acids, peptides, proteins, carbohydrates,lipids and the like by utilizing metabolic systems of cells includingenergy metabolic systems has been performed for a long time. However, inconventional metabolically controlled fermentation, fermentation systemfor the objective product is constructed through trials on screening andconcentration of variants resistant to an analogue having a structureanalogous to an objective product. Further, to control the constructedfermentation system, physical factors such as pH and temperature andchemical factors such as substrate concentration and addition ofinducers must be changed. Therefore, various regulatory mechanisms ofthe living body are often affected, and many parameters must bedetermined for optimization of the production scheme. Moreover,regulatory methods of this type have a drawback that it takes a longperiod of time to obtain a reaction to stimulation. Therefore, if it ispossible construct a method for controlling metabolism which enablesproper turning on and turning off a desired intracellular metabolicsystem so as to produce a desired product in a necessary amount when itis required, it will greatly contribute to fermentation processes.

[0005] Accordingly, the present inventors studied out a method forartificially controlling metabolism of cells including energy metabolismby utilizing the peptide stably binding a non-heme iron, which is thephotoreactive site of NHase mentioned above. That is, such a peptide asmentioned above is introduced into a protein which functions in ametabolic system of an objective product to impart photoreactivity tothe protein so that the metabolic system can be controlled by presenceor absence of light irradiation. In this method, photo-control ofactivity is realized by modifying a protein which functions in aspecific reaction system in a metabolic pathway of an objective product.Because the protein to be modified can be arbitrarily selected dependingon the purpose, the method has advantages that the screening step bytrial and error like in the conventional method does not required, andthat fermentation system of which metabolism is controlled can beprecisely constructed. Furthermore, because activation of enzyme isachieved by photostimulation, fermentation operation can be performedmore simply and quickly compared with the conventional methods.

[0006] However, when such a peptide sequence as mentioned above isintroduced into various kinds of proteins working in intracellularsubstance metabolic systems or energy metabolic systems to impartphotoreactivity to the metabolism or the energy metabolism, a relativelylarge peptide like the aforementioned peptide of 24 residues might haveproblems. The problems are that efficient reaction could not beobtained, or introduction of the peptide impairs the original functionof the labeled protein in high possibility, because the relatively largeprotein contains a large fraction of sequence other than the minimumportion essential for the photoreaction.

SUMMARY OF THE INVENTION

[0007] Therefore, the object of the present invention is to provide apeptide sequence capable of efficient photoreaction, which is a peptidechain of a minimum unit capable of imparting photoreactivity, not likelyto impair an original function of a labeled protein, and a method forenabling control of metabolic reaction by utilizing the peptide toimpart photoreactivity to cells.

[0008] The present invention relates to a peptide having a sequencerepresented by the following general formula (1).

X₁X₂C₁X₃X₄C₂SC₃X₅X₆X₇  (1)

[0009] wherein X₁ to X₇ represents an arbitrary amino acid, C₁represents cysteine, C₂ represents cysteinesulfinic acid, C₃ representscysteinesulfenic acid, and S represents serine.

[0010] An embodiment of the present invention is the aforementionedpeptide wherein the sequence represented by the general formula (1) isrepresented as IVC₁SLC₂SC₃TAW wherein I represents isoleucine, Vrepresents valine, C₁ represents cysteine, C₂ representscysteinesulfinic acid, C₃ represents cysteinesulfenic acid, S representsserine, L represents leucine, T represents threonine, A representsalanine, and W represents tryptophan.

[0011] Another embodiment of the present invention is the aforementionedpeptide wherein X₁ to X₇ in the sequence represented by the generalformula (1) are selected from amino acids which can maintain higherorder structure of a peptide represented by IVC₁SLC₂SC₃TAW in nitrilehydratase derived from Rhodococcus sp.N-771.

[0012] A further embodiment of the present invention is a peptide havinga sequence represented by the following general formula (2):

C₁X₃X₄C₂SC₃  (2)

[0013] wherein X₃ and X₄ represent arbitrary amino acids, C₁ representscysteine, C₂ represents cysteinesulfinic acid, C₃ representscysteinesulfenic acid, and S represents serine. Examples of the abovepeptide include, for example, the aforementioned peptide wherein thesequence represented by the general formula (2) is represented asC₁SLC₂SC₃ wherein C₁ represents cysteine, C₂ represents cysteinesulfinicacid, C₃ represents cysteinesulfenic acid, S represents serine, and Lrepresents leucine. Examples of the above peptide further include, forexample, the aforementioned peptide wherein X₃ and X₄ in the sequencerepresented by the general formula (2) are selected from amino acidswhich can maintain higher order structure of a peptide represented byC₁SLC₂SC₃ in nitrile hydratase derived from Rhodococcus sp. N-771.

[0014] The peptides of the present invention can be a peptide which canimpart photoreactivity to a protein by binding a non-heme iron, or apeptide which can form a claw setting structure by binding a non-hemeiron.

[0015] The present invention also relates to a method for impartingphotoreactivity to a cell by introducing one of the aforementionedpeptide sequences of the present invention into at least one proteinwhich is involved in a metabolic system and/or energy metabolic systemof the cell.

[0016] In the method of the present invention, together with the peptidesequence, a non-heme iron binding to the peptide can be introduced.

[0017] The present invention also relates to a cell having an NOproducing system and photoreactivity wherein one of the aforementionedpeptide sequences of the present invention is introduced into at leastone protein which is involved in a metabolic system and/or energymetabolic system of the cell.

[0018] The cell of the present invention may be, for example, a cellintroduced with the peptide sequence and a non-heme iron binding to thepeptide, or a cell wherein the peptide sequence and the non-heme ironform a claw setting structure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 represents results of reverse phase HPLC of products fromdigestion with thermolysin, carboxypeptidase-Y and leucineaminopeptidase-M.

[0020]FIG. 2 is a UV absorption spectrum of a peak at retention time of11.6 minutes in reverse phase HPLC of products from the digestion withthermolysin, carboxypeptidase-Y and leucine aminopeptidase-M.

[0021]FIG. 3 shows a claw setting structure formed by a peptideC₁SLC₂SC₃ binding to a non-heme iron. In this figure, a binding NOmolecule is also shown.

[0022]FIG. 4 shows a hetero-tetramer structure ((αβ)₂) in an asymmetricunit.

[0023]FIG. 5 shows omit-annealed Fo-Fc maps of an active site region.

[0024]FIG. 6 shows a claw setting structure formed by a peptideC₁SLC₂SC₃ binding to a non-heme iron.

[0025]FIG. 7 shows an electrospray mass spectrum.

DETAILED DESCRIPTION OF THE INVENTION

[0026] As described above, by enzymatically degrading with trypsin the αsubunit of NHase having photoreactivity similar to that of wild typeNHase and binding an NO-binding type non-heme iron center, a peptidecomplex consisting of 24 residues of ₁₀₅N to ₁₂₈K which is binding oneiron atom and NO per one peptide can be isolated. The present inventorsfurther digested the above peptide of 24 residues with thermolysin,leucine aminopeptidase-M and carboxypeptidase-Y, and found a peptidewhich is composed of 11 residues, forms a stable complex with a non-hemeiron, and is a minimum unit for imparting photoreactivity. This peptideis a peptide represented as IVC₁SLC₂SC₃TAW wherein I representsisoleucine, V represents valine, C₁ represents cysteine, C₂ representscysteinesulfinic acid, C₃ represents cysteinesulfenic acid, S representsserine, L represents leucine, T represents threonine, A representsalanine, and W represents tryptophan.

[0027] The present inventors further examined the structure of theactive center of the above peptide by X-ray crystallographic analysis.As a result, it was found that the 6-residue C₁SLC₂SC₃ containing C₁,C₂, S and C₃ formed a stable complex directly with the non-heme iron.

[0028] Mass spectrometry analysis was performed for the 24-residuepeptide in order to obtain its microstructural information. When mass ofthe peptide was determined under light irradiation, the obtained masswas larger than its theoretical molecular weight by 32 Da. All of theamino acid residues of this sequence except for the three cysteineresidues had been determined by analysis with a protein sequencer.Therefore, the peptide was subjected to reduction and thencarboxymethylation, and analyzed by the protein sequencer. As a result,only the 112nd cysteine was not carboxymethylated. Therefore, theobtained peptide was subjected to second digestion with thermolysin, andthen degraded with aminopeptidase for amino acid analysis. The 112ndcysteine was detected as the same peak as commercially availablecysteinesulfinic acid, and thus it was confirmed that this residueexisted as sulfinic acid. In contrast, any cysteine residue was notmodified in an α, subunit which was expressed as a recombinant in E.coli, and it has been revealed that the 112nd cysteine is specificallymodified to sulfinic acid in Rhodococcus sp. N-771.

[0029] In the aforementioned peptide, C₃ represents cysteinesulfenicacid, and it was confirmed that C₃ is cysteinesulfenic acid by analysisof the peptide digested with trypsin using Fourier transform ioncyclotron resonance mass spectrometry (FT-ICR MS).

[0030] The present invention further relates to a peptide having asequence represented by the general formula (1). This peptide wasdefined based on the aforementioned sequence IVC₁SLC₂SC₃TAW with aconsideration of the binding of this peptide and iron atom.

X₁X₂C₁X₃X₄C₂X₅X₆X₇  (1)

[0031] In the formula, X₁ to X₇ represent arbitrary amino acids, but inparticular they are suitably selected from amino acids which canmaintain the higher order structure of the above sequenceIVC₁SLC₂SC₃TAW.

[0032] The present invention also relates to a peptide having a sequencerepresented by the general formula (2). This peptide was defined basedon the aforementioned sequence C₁SLC₂SC₃ with a consideration of thebinding of this peptide and iron atom.

C₁X₃X₄C₂SC₃  (2)

[0033] In the formula, X₃ and X₄ represent arbitrary amino acids, but inparticular they are suitably selected from amino acids which canmaintain the higher order structure of the above sequence C₁SLC₂SC₃.

[0034] In particular, the above sequences IVC₁SLC₂SC₃TAW and C₁SLC₂SC₃are bound to a non-heme iron to form the claw setting structure with itrepresented in FIG. 3. C₁(Cys 109), C2 (Cys-SO₂H 112), S (Ser 113) andC₃ (Cys-SOH 114) coordinate to the non-heme iron. In the figure, a boundNO molecule is also represented.

[0035] Accordingly, X₁ to X₆ in the sequence represented by the generalformula (1), and X₃ and X₄, in the sequence represented by the generalformula (2) are suitably selected from amino acids which can bind to thenon-heme iron to form the claw setting structure.

[0036] The peptide of the present invention can be obtained by, forexample, enzymatical degradayion wild type NHase derived frommicroorganisms such as Rhodococcus sp. N-771 (FERM P-4445). First, bytreating α subunit of wild type NHase with trypsin, the 24-residuepeptide can be obtained. Then, by successively digesting this peptidewith thermolysin, then leucine aminopeptidase and carboxypeptidase M,the objective sequence IVC₁SLC₂SC₃TAW can be obtained.

[0037] Alternatively, the peptide can be produced by a publicly knowngenetic engineering technique based on the desired amino acid sequence.

[0038] The present invention includes a method for impartingphotoreactivity to cells by introducing the aforementioned peptidesequence of the present invention into at least one protein which isinvolved in a metabolic system and/or energy metabolic system of thecells. By introducing the above peptide sequence of the presentinvention into a protein, a photoreactive non-heme iron center havingthe claw setting, structure shown in FIG. 3 can be formed. The presentinvention also includes a method for imparting photoreactivity to cellsby introducing the aforementioned peptide sequence of the presentinvention together with a non-heme iron which is binding to the peptideinto at least one protein which is involved in a metabolic system and/orenergy metabolic system of the cells. The aforementioned peptidesequence of the present invention can be introduced into one or severalkinds of proteins. In particular, by introducing it into several kindsof proteins, there can be obtained advantages that metabolic rates canbe controlled among several pathways, and a metabolic pathway suitablefor the desired production can be selectively activated.

[0039] The present invention further includes a cell having an NOproducing system and photoreactivity wherein the aforementioned peptidesequence of the present invention is introduced into at least oneprotein which is involved in a metabolic system and/or energy metabolicsystem of the cell. The present invention also includes a cell having anNO producing system and photoreactivity wherein the aforementionedpeptide sequence of the present invention is introduced together with anon-heme iron which is binding to the peptide (having the claw settingstructure shown in FIG. 3) into at least one protein which is involvedin a metabolic system and/or energy metabolic system of the cell. Thepeptide sequence of the above the present invention can be introducedinto one or several kinds of proteins. In particular, by introducing itinto several kinds of proteins, there can be obtained advantages thatmetabolic rates can be controlled among several pathways, and ametabolic pathway suitable for the desired production can be selectivelyactivated.

[0040] As the protein to which photoreactivity is imparted, for example,enzymes having a mononuclear non-heme iron such as catechol dioxygenase,lipoxygenase and tyrosine hydroxylase can be mentioned. When theseenzymes are used, genes for them are isolated to form a recombinantexpression vector, and then a nucleotide sequence to be translated intothe peptide of the present invention is introduced into a gene regionfor the portion around the non-heme iron binding site by a recombinantDNA technique to form a expression vector for a recombinant proteinhaving a photoreactive non-heme iron center.

[0041] When it is desired to impart photoreactivity to a specific stepof an enzymatic process involving several enzymes, impartingphotoreactivity may be achieved as follows. First, a recombinant genefor a protein functioning in the step to which photoreactivity isdesired to be imparted in which the photoreactive non-heme iron centeris introduced is constructed by the method described above. Then, therecombinant gene is transferred into a cell having production process ofa target biomolecule by using a known genetic engineering technique suchas homologous recombination to impart photoreactivity to the cell. Thus,photo-control of a production process of useful biomolecule can berealized.

[0042] A source of NO supply can be introduced by constructing anexpression vector for an NO synthase utilizing arginine as a substrate,and introducing it into a host concurrently with the aforementionedvector for expressing a mutant. Because the NO synthase is activatedwith calcium ion, inactivation by NO can be controlled by two kinds offactors, dark condition and calcium ion. Thus, a protein havingphotoreactivity can be prepared.

EXAMPLES

[0043] The present invention will be further explained with reference tothe following examples.

EXAMPLES

[0044] All manipulations were operated in the dark to avoidphotodissociation of NO from the iron center. The isolation of a subunitfrom NHase was performed according to the method described in J.Biochem., 119, 407-413 (1996).

[0045] The α subunit which had been isolated from inactive NHase derivedfrom Rhodococcus sp. N- 771 (FERM P-4445) was thoroughly desalted usinga Centriprep-10 (Amicon) with 20 mM Tris-HCl, pH 8.0, containing 10 mMCaCl₂ and 2 mM 2-mercaptoethanol. The α subunit at a final concentrationof 2-2.5 mg/ml in 500 μl was treated with TPCK trypsin (14 μg) for 3hours at 37° C. The digests were subjected to reversed-phase HPLC with aCapcell pak C18 column (4.6×250 mm, Shiseido). The elution conditionswere as follows. Solvent A was 20 mM Tris-HCl, pH 7.5, containing 2 mM2-mereaptoethanol, and Solvent B was 20% A+80% acetonitrile. The contentof B was increased as; 0-2 min, 0%; 2-4 min, 0-20%; 4-24 min, 20-60% ;and 24-26 min, 60-100%. The flow rate was 1.0 ml/min. The eluent wasmonitored by a diode array detector, HEWLETT PACKARD, HP1090 LiquidChromatography. Sequence of the product provided was determined by anamino acid sequencer. As a result, it was a single fragment composed of24 residues of ₁₀₅Asn-₁₂₈Lys[N-V-I-V-C-S-C-T-A-W-P-I-L-G-L-P-P-T-W-Y-K]. Solvent A was changed to 5mM ammonium acetate, pH 7.5, and reversed-phase HPLC was performed underthe same condition as described above. When the amount of iron atomsbound to in the resulted peptide was determined by ICP-MS, it was foundthat one peptide bound one Fe atom.

[0046] Then, the peptide resulted above was further digested. Thepeptide (1.35 μg) in 20 mM Tris-HCl, pH 7.5 was treated with 1 μg ofthermolysin for 1 hours at 37° C., and then with carboxypeptidase-Y (1μg) and leucine aminopeptidase-M (1 μg) for 12 hours at 37° C. Thedigests were separated by reversed-phase HPLC using Capcell pak C18column (4.6×250 mm, Shiseido). The gradient condition was as follows.Solvent C was 20 mM ammonium acetate, pH 7.5 and Solvent D was 20%Solvent C+80% acetonitrile. The column was equilibrated with 100% ofSolvent C and a linear gradient was run from 0% of solvent D to 80% overa period of 20 min at a flow rate of 0.2 ml/min. The result of thereversed-phase HPLC is shown in FIG. 1, and a UV absorption spectrum ofa peak at retention time of 11.6 minutes in the reversed-phase HPLC isshown in FIG. 2. In the UV absorption spectrum of the peak at retentiontime of 11.6 minutes, an absorption peak at 370 nm was observed, and itindicates that the contained fragment was associated with a nitrosylatediron. In a photo-irradiated peptide, the aforementioned absorption peakat 370 nm completely disappeared.

[0047] Sequence of the resulted product was determined by an amino acidsequencer. As a result, it was a single fragment composed of 11 residuesof ₁₀₇I-V-S-L-C-S-C-T-A-₁₁₇W.

[0048] Existence of Post-translational Modification

[0049] Cysteine residues cannot be detected when sequencing wasperformed with an amino acid sequencer. The aforementioned 24-residuepeptide of ₁₀₅N to ₁₂₈K (₁₀₅NVIVCSLCSCTAWPILGLPPTWYK₁₂₈) contains threecysteine residues, and the remaining 21 residues have already beenidentified. Therefore, also considering the result of the massspectrometry, the peptide fragment provided from trypsin digestion wassubjected to carboxymethylation after reduction to alkylate the cysteineresidues, and then the analysis by the amino acid sequencer wasattempted. As a result, among the cysteine cluster containing threecysteines (₁₀₉C-S-L-₁₁₂CS-₁₁₄C), the 112nd cysteine still could not bedetected as before, whereas the 109th and the 114th cysteines could bedetected as carboxymethylcysteines. At the same time, mass of thissample was measured by using MALDI-TOF MS (matrix-assisted laserdesorption ionization time of flight mass spectrometer) as a massspectrometer. As to the mass number, in addition to the molecular weightof ₁₀₅N to ₁₂₈K (2663.3), increment considered to be due to binding oftwo carboxymethyls (molecular weight: 59) and further increment of 32 Dawere detected.

[0050] Identification of Cysteinesulfinic Acid

[0051] From the results of mass spectrometry and amino acid sequencing,it became clear that modification of 32 Da increment occurred at the112nd cysteine. It is assumed that sulfur (32) or oxygen-molecule (16×2)corresponds to the mass number of 32. However, if it was due to sulfuratom (Cys-S₂H), it should be carboxymethylated after reduction.Therefore, the most probable modification was considered to be oxidationby two oxygen atoms. The present inventors considered that the cysteineresidue is present in the fragment as cysteinesulfinic acid (Cys-SO₂H),which corresponds to cysteine residue oxidized by two oxygen atoms, andidentified it by amino acid analysis as follows. Amino acid compositionanalysis was performed by the method established by Hayashi et al.wherein antipodes of amino acids are derivated with a fluorescentreagent, (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), and separated byreversed-phase chromatography (Hayashi et al., 1993).

[0052] In order to prevent the oxidation of cysteine residue, hydrolysisof the peptide was performed with an enzyme (leucine aminopeptidase).However, since the 112nd, amino acid is the eighth amino acid residuefrom the N-terminus of the peptide, and yield will be decreased as it ismore remote from the N-terminus, the fragment was preliminarilysubjected to secondary digestion with thermolysin. The resultingdigestion mixture was purified by reversed-phase chromatography toafford several fragments, and a fragment of the objective 8 residues offrom 111st leucine to 118th proline (₁₁₁L-X-S-CM-T-A-W-₁₁₈P wherein X isa residue considered to be cysteinesulfinic acid, and CM iscarboxymethylcysteine) was eluted at 7.8 min.

[0053] The molecular mass of the fragment obtained by the abovesecondary digestion was estimated by MALDI-TOF MS, and detected as970.7, which was larger than the theoretical molecular weight of 938.1by 32 Da. That is, the modification of 32 Da increment still remained inthis fragment. Then, it was hydrolyzed into individual amino acids withleucine aminopeptidase to perform amino acid composition analysis. Ascontrols, D,L-cysteic acid and L-cysteinesulfinic acid were analyzed,and respective elution time was 22.9 min (L-cysteic acid), 23.4 min(D-cysteic acid), and 26.7 min. From the area values of the peaks, itwas considered that about 39% of sulfinic acid was oxidized to cysteicacid in this measurement.

[0054] In the amino acid composition analysis of the peptide of 8residues obtained from the secondary digestion with thermolysin, peakswere detected at elution times of 22.7 min and 26.2 min. From the ratioof the area values, it was thought that about 56% of the residue wasconverted into cysteic acid. The yield of leucine of the N-terminus ofthis fragment was 97.3 pmol, whereas the yield of cysteinesulfinic acidincluding those oxidized into cysteic acid was 99.0 pmol(cysteinesulfinic acid: 44 pmol, cysteic acid: 55 pmol).

[0055] From the results mentioned above, it has been revealed that the112nd cysteine residue in the above peptide fragment was modified byoxidation with two oxygen atoms into cysteinesulfinic acid (Cys-SO₂H).

[0056] Structure Determination of the Nitrosylated NHase

[0057] The crystal of the nitrosylated NHase (inactive NHase) diffractedX-ray up to high resolution, 1.7 Å resolution, and belonged to the spacegroup of P2₁2₁2 with cell dimensions of a=117.4 Å, b=145.6 Å and c=52.1Å . Two αβ hetero-dimers existed in an asymmetric unit of the crystal.The crystal structure of the inactive NHase was determined by multipleisomorphous replacement (MIR) analysis followed by density modificationand non-crystallographic symmetry averaging. The structural model wascrystallographically refined at 1.7 Å resolution (R-factor 0.179,R-_(free)0.228). FIG. 4 shows a hetero-tetramer structure ((αβ)₂) in anasymmetric unit. The folding pattern of the inactive NHase is verysimilar to that of the photoactivated enzyme, showing that theconformation is conserved between the inactive and photoactivatedenzymes. While the inactive NHase formed a hetero-tetramer in thecrystal, sedimentation equilibrium and dynamical light scatteringmeasurements showed that the enzyme existed in a dimer-tetramerequilibrium in solution depending on its concentration. The non-hemeiron center was located at the interface between the two subunits in thehetero-dimer.

[0058] Structure of the Active Center

[0059]FIG. 5 shows the omit-annealed Fo-Fc maps of the active siteregion. Each residue was clearly resolved. The active site was composedof four amino acid residues from the α subunit (αCys¹⁰⁹, αCys¹¹²,αSer¹¹³, αCys¹¹⁴) and two amino acid residues from the β subunit(βArg⁵⁶, βArg¹⁴¹). The ligands to the non-heme iron atom are sulfuratoms of the three cysteine residues (αCys¹⁰⁹, αCys¹¹², αCys¹¹⁴), mainchain amide nitrogen atoms (αSer¹¹³, αCys¹¹⁴) and nitric oxide (NO).Only the site occupied by nitric oxide is accessible from solvent. Theatoms coordinating to the iron of the inactive type were identical tothose in the photoactivated one except for the nitrogen of nitric oxide.The two sulfur atoms (S γ atoms of αCys¹¹² and αCys¹¹⁴), the two amidenitrogen atoms and the iron atom were arranged in the same plane. Thelength of the Fe—N(NO) bond in the inactive NHase was 1.65 Å, which wascomparable to those in many nitrosyl iron(III) complexes. The nitricoxide coordinated with the non-heme iron(II) in a bent configurationwith a Fe—N—O angle of 158.6° tilted toward the middle of αCys¹¹² and aCys¹¹⁴.

[0060] Post-translational Modifications in the Active Center

[0061] General structure of the active center can be seen in theomit-annealed Fo-Fc maps. Extra electron densities appeared around theSγ atoms of a Cys¹¹² and α Cys¹¹⁴ (FIG. 5). Since αCys¹¹² ispost-translationally modified to a cysteinesulfinic acid (Cys-SO₂H), thetwo additional electron densities around S γ of αCys¹¹² is thought tocorrespond to the two oxygen atoms of the sulfinyl group. It was assumedthat αCys¹¹⁴ was also post-translationally modified, because there wasno atom to ascribe the additional electron density close to Sγ ofαCys¹¹⁴ in any known models. To confirm this idea, the tryptic digestsof the inactive NHase were examined by FT-ICR MS. FIG. 7a shows thepositive electrospray ionization mass spectra of a tryptic digest ofinactive NHase α subunit in the neutral condition. Two signals with m/zof 1347.65 and 1383.13 were assigned for doubly charged, (M+2H)²⁺, ironcenter peptides (αAsn¹⁰⁵-αLys¹²⁸) without and with Fe³⁺, respectively.The former contained α Cys¹⁰⁹-S-S-αCys¹¹⁴ and αCys¹¹²-SO₂H (M_(r),=2693.31), and the latter contained α Cys¹⁰⁹-S⁻, αCys¹¹⁴-SO⁻ andαCys¹¹²- SO²⁻ (M^(r)2764.23). Assuming no post-translationalmodification occurred on αCys¹¹⁴, expected M_(r) of the latter (2748.24)was smaller by 16.02 than the observed value. Upon addition of aceticacid, the latter signal disappeared and the relative intensity of theformer, was doubled or more (FIG. 7b). In the acidic condition, thepeptide released Fe³⁺, which was replaced by 3 protons, and a disulfidebond was formed between αCys-SOH¹¹⁴ and αCys-SH¹⁰⁹ as a result a watermolecule is produced. The measured mass difference of two signals(35.48×2=70.96) was well in accord with the calculated difference(55.93−3.03+18.01=70.92). Thus, we concluded that αCys¹¹⁴ is modified toCys-SOH, and the additional electron density corresponds to the oxygenatom of the sulfenyl group. It is considered that, since acidiccondition was used for the experiment on cysteinesulfenic acid, thepresence of acid-labile cysteinesulfenic acid was overlooked.

[0062] Stabilization of Nitric Oxide by “the Claw Setting”

[0063] As shown in FIG. 6, three oxygen atoms, O δ1 of αCys-SO₂H¹¹²O δof α Cys-SOH¹¹⁴ and O γ of αSer¹¹³, were protruded from the planecontaining the iron atom by 1.5 Å like claws (of rings), and a nitricoxide molecule was held at the center of the three “claws”. Thisstructure is named as “claw setting”. These three oxygen atoms arelocated at a distance from the nitric oxide molecule close enough tohave strong interaction within them. The nitrosyl iron complex of theNHase, whose iron exists in a low-spin ferric(II) state as indicated byESR studies, is stable more than one year under aerobic condition aslong as shielded from light. Moreover, the nitrosylated iron center isstable even in a proteolytic fragment from αIle¹⁰⁷ to a Trp¹¹⁷, whereasthe association constant of nitric oxide to ferric(III) irons are muchsmaller than those to ferrous(II) ones in hemeproteins, and theassociation is generally unstable in air. The extraordinary stability ofthe nitrosyl non-hem ferric(III) iron center in NHase having iron(III)is likely to be due to the interaction between the nitric oxide and theoxygen atoms in the “claw setting”. On the contrary, the nitric oxideimmediately dissociates from NHase after light irradiation. Fouriertransform infrared difference spectrum showed that a local structuralchange occurs around the iron center upon light irradiation. Theseresults suggest that light irradiation breaks the Fe—N(NO) bond, andthus weakens the interaction between the oxygen atoms and the nitricoxide which induces the structural change of the “claw setting”. Takentogether, the photoreactivity of Fe-type NHase is likely to be explainedin terms of the “claw setting”.

[0064] αCys-SO₂H¹¹² and αCys-SOH¹¹⁴ were stabilized by hydrogen bondsformed with βArg⁵⁶ and βArg¹⁴¹ (FIG. 6). These arginine residues areconserved in all known NHases. The replacement of these residues withother amino acids resulted in the loss of activity, and induced asignificant change in the absorption spectra reflecting the electronicstate of the catalytic center (M. Tsujimura et al.). This fact suggeststhat the “claw setting” is also important for the enzymatic activity ofNHase.

[0065] According to the present invention, there can be provided apeptide with a shorter peptide chain capable of impartingphotoreactivity, which can be easily introduced into a protein with alittle risk in degrading original function of the protein. It is also bepossible to impart photoreactivity to a cell by utilizing this peptide.As a result, it becomes possible to control production of a great numberof useful biomolecules such as amino acids, peptides, proteins,carbohydrates, and lipids by presence and absence of light irradiation.

Sequence Listing

[0066] INFORMATION FOR SEQ ID NO:1:

[0067] LENGTH OF SEQUENCE: 11 amino acids

[0068] TYPE OF SEQUENCE: amino acid

[0069] TOPOLOGY: linear

[0070] TYPE OF SEQUENCE: peptide

[0071] SEQUENCE DESCRIPTION:

[0072] Ile Val Cys Leu Xaa Ser Cys Thr Ala Trp

[0073] INFORMATION FOR SEQ ID NO:2:

[0074] LENGTH OF SEQUENCE: 5 amino acids

[0075] TYPE OF SEQUENCE: amino acid

[0076] TOPOLOGY: linear

[0077] MOLECULE TYPE OF SEQUENCE: peptide

[0078] SEQUENCE DESCRIPTION:

[0079] Cys Leu Xaa Ser Cys

1 6 1 10 PRT Rhodococcus sp. N-771 PEPTIDE (5) Amino acid at position 5is Xaa wherein Xaa = an arbitrary amino acid. 1 Ile Val Cys Leu Xaa SerCys Thr Ala Trp 1 5 10 2 5 PRT Rhodococcus sp. N-771 PEPTIDE (3) Aminoacid at position 3 is Xaa wherein Xaa = an arbitrary amino acid. 2 CysLeu Xaa Ser Cys 1 5 3 11 PRT Rhodococcus sp. N-771 PEPTIDE (1)..(11)Amino acids at positions 1, 2, 4, 5, 9, 10 & 11 are Xaa wherein Xaa = anarbitrary amino acid. 3 Xaa Xaa Cys Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa 1 510 4 11 PRT Rhodococcus sp. N-771 PEPTIDE (1)..(11) Amino acid atposition 6 is Xaa wherein Xaa = cysteinesulfinic acid. 4 Ile Val Cys SerLeu Xaa Ser Xaa Thr Ala Trp 1 5 10 5 21 PRT Rhodococcus sp. N-771 5 AsnVal Ile Val Cys Ser Cys Thr Ala Trp Pro Ile Leu Gly Leu Pro 1 5 10 15Pro Thr Trp Tyr Lys 20 6 10 PRT Rhodococcus sp. N-771 6 Ile Val Ser LeuCys Ser Cys Thr Ala Trp 1 5 10

1. A peptide having a sequence represented by the following generalformula (1): X₁X₂C₁X₃X₄C₂SC₃X₅X₆X₇  (1) wherein X₁ to X₇ represents anarbitrary amino acid, C₁ represent cysteine, C₂ representscysteinesulfinic acid, C₃ represent cysteinesulfenic acid, and Srepresents serine.
 2. The peptide of claim 1 wherein the sequencerepresented by the general formula (1) is represented as IVC₁SLC₂SC₃TAWwherein I represents isoleucine, V represents valine, C₁ representscysteine, C₂ represents cysteinesulfinic acid, C₃ representscysteinesulfenic acid, S represents serine, L represents leucine, Trepresents threonine, A represents alanine, and W represents tryptophan.3. The peptide of claim 1 wherein X₁ to X₇ in the sequence representedby the general formula (1) are selected from amino acids which canmaintain higher order structure of peptide represented by IVC₁SLC₂SC₃TAWmentioned in claim 2 in nitrile hydratase derived from Rhodococcus sp.N-771.
 4. A peptide having a sequence represented by the followinggeneral formula (2): C₁X₃X₄C₂SC₃  (2) wherein X₃ and X₄ representarbitrary amino acids, C₁ represents cysteine, C₂ representscysteinesulfinic acid, C₃ represents cysteinesulfenic acid, and Srepresents serine.
 5. The peptide of claim 4 wherein the sequencerepresented by the general formula (2) is represented as C₁SLC₂SC₃wherein C₁ represents cysteine, C₂ represents cysteinesulfinic acid, C₃represents cysteinesulfenic acid, S represents serine, and L representsleucine.
 6. The peptide of claim 4 wherein X₃ and X₄ in the sequencerepresented by the general formula (2) are selected from amino acidswhich can maintain higher order structure of a peptide represented byC₁SLC₂SC₃ mentioned in claim 5 in nitrile hydratase derived fromRhodococcus sp. N-771.
 7. The peptide of claim 1 which can impartphotoreactivity to a protein by binding to a non-heme iron.
 8. Thepeptide of claim 4 which can impart photoreactivity to a protein bybinding to a non-heme iron.
 9. The peptide of claim 1 which can form aclaw setting structure by binding to a non-heme iron.
 10. The peptide ofclaim 4 which can form a claw setting structure by binding to a non-hemeiron.
 11. A method for imparting photoreactivity to a cell byintroducing the peptide sequence of claim 1 into at least one proteinwhich is involved in a metabolic system and/or energy metabolic systemof the cell.
 12. A method for imparting photoreactivity to a cell byintroducing the peptide sequence of claim 4 into at least one proteinwhich is involved in a metabolic system and/or energy metabolic systemof the cell.
 13. The method of claim 11 wherein a non-heme iron bindingto the peptide is introduced with the peptide sequence.
 14. The methodof claim 12 wherein a non-heme iron binding to the peptide is introducedwith the peptide sequence.
 15. A cell having an NO producing system andphotoreactivity wherein the peptide sequence of claim 1 is introducedinto at least one protein which is involved in a metabolic system and/orenergy metabolic system of the cell.
 16. A cell having an NO producingsystem and photoreactivity wherein the peptide sequence of claim 4 isintroduced into at least one protein which is involved in a metabolicsystem and/or energy metabolic system of the cell.
 17. The cell of claim15 which is introduced with a non-heme iron binding to the peptidetogether with the peptide sequence.
 18. The cell of claim 16 which isintroduced with a non-heme iron binding to the peptide together with thepeptide sequence.
 19. The cell of claim 17 wherein the peptide sequenceand the non-heme iron form a claw setting structure.
 20. The cell ofclaim 18 wherein the peptide sequence and the non-heme iron form a clawsetting structure.